phiC31 integrase for recombination-mediated single-copy insertion and genome manipulation in Caenorhabditis elegans
Journal
Genetics
Journal Volume
220
Journal Issue
2
Date Issued
2022-02-04
Author(s)
Yang, Fang-Jung
Chen, Chiao-Nung
Chang, Tiffany
Cheng, Ting-Wei
Chang, Ni-Chen
Kao, Chia-Yi
Lee, Chih-Chi
Huang, Yu-Ching
Hsu, Jung-Chen
Li, Jengyi
Lu, Meiyeh J
Wang, John
Abstract
Caenorhabditis elegans benefits from a large set of tools for genome manipulation. Yet, the precise single-copy insertion of very large DNA constructs (>10 kb) and the generation of inversions are still challenging. Here, we adapted the phiC31 integrase system for C. elegans. We generated an integrated phiC31 integrase expressing strain flanked by attP sites that serves as a landing pad for integration of transgenes by recombination-mediated cassette exchange (RCME). This strain is unc-119(-) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations were obtained for insert sizes up to ∼33.4 kb. Taking advantage of this integration method we establish a dual-color fluorescent operon reporter system able to study post-transcriptional regulation of mRNA. Last, we show that large chromosomal segments can be inverted using phiC31 integrase. Thus, the phiC31 integrase system should be a useful addition to the C. elegans toolkit.
Subjects
dual-fluorescent operon reporter; integration; inversion; phiC31 integrase; recombination-mediated cassette exchange
Type
journal article